Resistance to drugs of microbes poses an increasing problem to the health care system. Resistant microbes, such as bacteria and unicellular fungi, but also viruses, may complicate the treatments of infections in critically ill patients, especially in surgery, hemato-oncology and intensive care in general. Microbial isolates that are resistant to more than one, and even to all available drugs are also known. Especially multi-resistant bacteria that are resistant to almost all antibiotics are a problem. It is therefore important to have a fast and accurate method to establish whether a microbe is resistant to a drug and even more important to which kind of drug, this is especially the case for antibiotic resistant bacteria. A quick and accurate determination of the kind of resistance is vital for the choice of antimicrobial therapy. It is also important to treat the microbes as specifically as possible to avoid further development of resistance. Therefore, routine microbial laboratories need fast and reliable methods to detect resistance in microorganism and establish the kind and preferably also the degree of resistance against a drug.
Most current techniques for susceptibility determinations of microorganisms are laborious and time-consuming as they encompass the growing of the microorganism. Also genetic tests are available to detect known resistance genes in the genome of the pathogen. The drawbacks of the genetic testing is that one will only find what one is looking for, as specific primers are required for known resistance genes. If for another resistance, no primers are used or available, this resistance gene will not be identified. Furthermore, a resistance gene may be present, but may not be expressed or to a low level. This will lead to a positive identification of resistance while the microorganism is not resistant e.g. the gene for a chromosomal encoded AmpC β-lactamase in E. coli may be present but the microorganism may still be susceptible to ampicillin because the gene is not expressed or only expressed to a low level.
Mass spectrometry has been used to identify and type microorganism. In recent years MALDI-TOF MS has been used to measure protein profiles of microorganism. Mass spectra are acquired and compared to a database of reference mass spectra of microorganisms where after software-assisted search the identification of microorganism is determined (U.S. Pat. No. 8,293,496). The advantages of such a system are fast determination of the microorganism, i.e. approximately 2 minutes vs 6-8 hours with phenotypical determination, and whole microorganism may be used so no need for further purification steps. About 84%-95% of clinical isolates tested were identified correctly with MALDI-TOF MS (Eigner et al (2009) din Lab 55(7-8):289-296; Seng et al (2009) Clin Infect Dis 49(4):543-551). The drawback is that in the range of measurement, usually between 3-11 kDa, ribosomal protein are predominantly identified, and the resistance may not relate directly to ribosomal proteins. In addition, with the MALDI-TOF techniques of the prior art, mutations in drug target proteins may not be detected. In addition, with the MALDI-TOF technique as used in the prior art, one is dependent on reference spectra, if no reference spectrum is present in the database, then the microorganism cannot be identified or will be wrongly identified. The strength of the method therefore depends on the quality of the database. Furthermore, with the current MALDI-TOF technique, one cannot distinguish between different drug conferring proteins within the same class. For instance, one may determine the presence of a beta-lactamase, but not which kind of beta-lactamase. In view of the fact that more than 900 different kinds of beta-lactamases are known, it may not be sufficient if one does not know which kind of beta-lactamase is present.
There is a need to optimize the detection of drug resistant microorganisms in a fast and reliable way. There is also a need to specifically identify the type of resistance, or even to identify the mutation conferring resistance or expression level of the resistance conferring protein.